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Biomedical subjects

W J Lindblad

Publications and source records attributed to W J Lindblad.

At least 37 records · Page 2Linked to original sources
PubMed1984

Glucocorticoids suppress formation of collagen by the hepatocyte. Studies in primary monolayer cultures of parenchymal cells prepared from adult rat liver.

We have established previously that the adult rat hepatocyte produces collagen under conditions of primary monolayer culture or under pathologic conditions in vivo. During the initial 3 days of incubation of hepatocytes in serum-free culture medium, collagen synthesis represented only 0.01%-0.05% of the synthesis of noncollagen proteins. Thereafter, rates of collagen synthesis rose dramatically, and after 9 days of incubation were increased an average tenfold in cultures derived from normal hepatocytes and 30-fold in cultures derived from regenerated liver. The spontaneous increase in the rates of collagen synthesis was almost completely blocked (94%) when cultures were incubated for 8 days in medium containing the synthetic glucocorticoid dexamethasone (10(-7) M). Dexamethasone was without effect on the synthesis of noncollagen proteins. Inhibition of collagen synthesis by dexamethasone exhibited a sigmoidal dose-response curve with 50% inhibition observed at a concentration of 10(-9) M. Collagen synthesis was diminished similarly by other glucocorticoids (hydrocortisone, methylprednisolone, triamcinolone, prednisone, all 10(-6) M) but not by other hormonal or nonhormonal steroids (progesterone, estradiol, testosterone, all 10(-5) M). We excluded increased collagen degradation as an explanation for these results by demonstrating that dexamethasone failed to increase the incorporation of radioactive proline into trichloroacetic acid-soluble radioactive hydroxyproline. Exposure of hepatocytes to dexamethasone during the first 48 h of incubation produced inhibition of collagen synthesis in 9-day-old cultures, even though washout studies with [3H]dexamethasone revealed negligible retention of the steroid by the cells. This observation suggests that among the initial, culture-adaptive responses of the hepatocyte are glucocorticoid-sensitive events that culminate in increased rates of collagen synthesis days later. We conclude that glucocorticoids may serve as natural suppressors of the potential of the hepatocyte to produce collagen.

Animals↗
PubMed1982

Mechanism for the differential induction of mutation by S9 activated benzo[a]pyrene employing either a glucose-6-phosphate-dependent NADPH-regenerating system or an isocitrate-dependent system.

A significant difference in mutation frequency has been observed in CHO cells exposed to benzo[a]pyrene with alternative activation systems. Each system employed rat-liver S9 homogenate with one using isocitrate dehydrogenase to provide reduced NADP, while the other method uses glucose-6-phosphate dehydrogenase. Total aryl hydrocarbon hydroxylase (AHH) activity was greater for the isocitrate dependent system, however, this yielded a lower level of HGPRT mutants. It was ascertained that this reduced mutation frequency may result from sequestering of B[a]P substrate by crystals in the medium, possibly calcium phosphate, which decreased the effective substrate concentration. This sequestration enhances B[a]P internalization, which would explain the dichotomy between the AHH values and the mutation frequency data. The production of specific B[a]P metabolites was also examined by reverse phase HPLC quantitation of extracts of solutions in which the two activation systems were used. The levels of 7,8 dihydroxybenzo[a]pyrene produced by the glucose-6-phosphate protocol were consistently greater than with isocitrate. This may also be a contributing mechanism for elevating the mutation frequency with this procedure. These results demonstrate several interactions between test compound, cells, and metabolizing system which must be considered with in vitro activation systems.

Animals↗
PubMed1982

An improved assay of mammalian collagenase activity, and its use to determine hepatic extracellular matrix susceptibility to degradation.

This rapid, sensitive method of determining collagenase (EC 3.4.24.7) activity incorporates several advantages of previous methods. Soluble [14C]acetylated collagen is prepared as the enzyme substrate and collagen-cleavage products are separated from noncleaved collagen by precipitation with dioxane/methanol. The assay is more reproducible than previous methods and has a lower detection limit, 15 mU of enzyme activity. We used the method in a competitive substrate assay with isolated extracellular hepatic matrix from cirrhotic and normal rat liver. Purified collagenase was consistently bound to normal rat matrix to a greater extent than to cirrhotic matrix, suggesting that in hepatic fibrosis the extracellular matrix is not as susceptible to collagenase degradation as that in normal liver.

Animals↗
PubMed1981

An improved method for the determination of aryl-4-hydroxylase activity.

An improved method for separation of tritiated benzo[a]pyrene from tritiated water in the aryl-4-hydroxylase assay is presented. Quantitative retention of benzo[a]pyrene was obtained with mixed cellulose acetate and nitrate filters. Enzymatic activities obtained with this modification agreed well with those determined using the standard charcoal column extraction. This modification greatly reduces the time required to perform this assay, and allows for the determination of enzyme activity in a large number of samples.

Animals↗
PubMedDate not supplied

Hepatic collagenase activity during carbon tetrachloride induced fibrosis.

The contribution of collagen degradation, as measured by collagenase activity, to the accumulation of collagen during hepatotoxic fibrogenesis was examined using a carbon tetrachloride rat model. Both active and inactive enzyme forms were determined with the inactive form quantitated following activation by limited trypsin digestion of the liver homogenate. The rate of collagen biosynthesis was monitored by quantitating hepatic prolyl hydroxylase activity and accumulation of collagen. In one study CCI4 was administered twice weekly, i.p. (0.2 mL, 33% v/v in light mineral oil) for sixteen weeks, with animals sacrificed every two weeks. Histologic examination of liver sections and serum alanine transaminase levels indicated a progressive necrosis and fibrosis which was confirmed by the increase in hepatic hydroxyproline content from 0.303 to 4.84 micrograms/mg wet wt. tissue. Prolyl hydroxylase activity increased in a time dependent manner to a maximum of 3.7 X control. This increase was accompanied by a large increase in both active collagenase (15.0-40.8 mU/g protein) and latent collagenase activity (69.8-191.7 mU/mg protein). These increases were maintained through the transition to irreversible fibrosis. The increase in active collagenase activity was positively correlated with collagen content. A second short term CCI4 treatment study confirmed a transient alteration in the active to latent collagenase ratio early in the fibrotic process. These results demonstrate the dynamic changes in hepatic collagenase levels and indicate that the fibrotic lesion is not dependent on decreased collagenase levels for collagen accumulation.

Animals↗
PubMedDate not supplied

A late defect in wound healing associated with recurrent spontaneous pneumothoraces: a presumptive role for abnormal collagenase activity.

This case report concerns an individual with a defect in wound healing which resulted in recurring, bilateral pneumothoraces during the late postoperative period. This patient had no history of systemic disease or wound healing abnormalities before his recurrent wound disruption. Physical examination and routine biochemical studies failed to identify any causative agent for the multiple wound dehiscences in the patient. Histologic examination of scar tissue showed collagen fiber bundles with a diameter 50% less than that of normal fibrils. Elastic fibers were barely visible, and the scar tissue included a large number of inflammatory cells. A significant finding was an elevated and aberrant expression of collagenase by a fibroblast cell line established from a skin biopsy specimen. This enhanced level of collagenase expression could be inhibited by treatment of the cells with diphenylhydantoin, an inhibitor of collagenase biosynthesis. After initiation of diphenylhydantoin therapy, the patient's scar formation normalized with the recurrent pneumothoraces. These findings support the conclusion that an abnormal expression of collagenase resulted in enhanced degradation of collagen in the patient's wounds, thereby leading to wound dehiscence.

Journal Article↗
PubMedDate not supplied

Altered collagen metabolism and delayed healing in a novel model of ischemic wounds.

Cellular mechanisms occurring in the healing wound have been well described in various animal models. However, the events associated with wound healing seen in ischemic skin have not been as thoroughly defined. In this series of experiments, we created a novel model of excisional skin wounds under gradient ischemia to study the cellular and extracellular events leading to delayed healing. We hypothesized that altered collagen metabolism accounts for delayed wound healing in ischemic skin. Three pairs of 4 mm punch wounds were made 4 days after bipedicle skin flaps were created on the dorsum of rats. Sham-operated control animals had the same punch wounds without flap creation. The kinetics of excisional wound healing were measured by means of computerized planimetry. In addition, wounds were excised with a 6 mm trephine, radiolabelled with ((3)H)-proline and in vitro collagen synthesis determined as collagenase digestible protein along with quantitation of DNA content. Total collagen deposition was determined as 4-hydroxy-L-proline by high-performance liquid chromatography, and wounds were histologically evaluated. Data was analyzed by means of two-way analysis of variance. Although control wounds healed by day 10, flap wounds consistently had greater surface area on days 2, 4, 6, 8, and 12 (p < 0.001). Relative collagen synthesis (% collagen/noncollagen protein), as measured by an in vitro synthesis method, showed no statistically significant differences between flap and controls wounds. However, the total collagen content (deposition) as measured by 4-hydroxy-l-proline was significantly lower in flap wounds compared with controls on days 7 (p < 0.05) and 9 (p < 0.001). In addition, a significant increase occurred in DNA content in the flap wounds on days 7 (p < 0.05) and 9 (p < 0.001) versus control wounds. These data indicate that, in ischemic wounds, significantly less collagen is deposited despite the inherent ability of the tissue to synthesize appropriate levels of collagen. Because the in vitro collagen synthesis technique only assesses the ability of the tissue to synthesize collagen in a well oxygenated environment, one cannot be assured that the tissue expresses this potential in vivo. However, these data are consistent with the hypothesis that the delay in wound closure is due to an alteration in collagen metabolism which results in a net decrease in collagen accumulation. Because of the observed increase in DNA within the ischemic wounds, we suggest that there is prolonged inflammation in these wounds which may enhance collagen degradation through the release of proteases. In addition, there may be an inability of the tissue to maintain appropriate levels of collagen in this inflammatory wound environment.

Journal Article↗
PubMedDate not supplied

Analysis of the effects of chitosan on inflammation, angiogenesis, fibroplasia, and collagen deposition in polyvinyl alcohol sponge implants in rat wounds.

Chitosan is a large molecular weight, positively charged polysaccharide extracted and purified from the chitin of crab shells. This compound has been shown to have hemostatic activity and has been suggested for use as a topical agent in tissue repair. The objective of this study was to analyze the effect of chitosan on the wound healing response to a standardized injury in the rat. The polyvinyl alcohol sponge implant model was used as a means to deliver either chitosan or its vehicle to a standardized subcutaneous wound on the backs of Sprague-Dawley rats. On days 8 and 14, the chitosan-treated implants contained primarily polymorphonuclear leukocytes compared with the vehicle controls which contained mainly macrophages, fibroblasts, collagen, and new blood vessels. High-performance liquid chromatography analysis for hydroxy-L-proline deposited in the sponge implants showed significantly lower amounts on both days 8 and 14 in the chitosan treatment group. These histologic and biochemical studies suggest that chitosan modulates wound healing by first reducing the influx of activated tissue macrophages, which in turn reduces the subsequent events of angiogenesis, fibroplasia, and connective tissue deposition.

Journal Article↗
PubMedDate not supplied

Perspective article: collagen expression by novel cell populations in the dermal wound environment.

Numerous collagenous structures must be reconstituted following injury to the skin in order to return function to this tissue. The basement membrane zone, vascular basement membranes, and the dense connective tissue of the dermis are examples of structures that contain a number of different collagen types and that may need replacement following injury. In addition, a scar is deposited at the site of damage in order to substitute for elements lost in the trauma and for elements that cannot be successfully replaced. Clearly, cells resident within the different compartments of the skin are able to synthesize and deposit collagen to reform these multiple structures. However, accumulating experimental evidence suggests that in addition to these resident cells, blood-borne cells may be responsible for the deposition of a portion of the newly synthesized collagen. Studies from this laboratory point to the activated monocyte as a potential source of collagen in the wound environment. Given the dynamics of the process, the hypothesis is proposed that during normal wound healing, the activated monocyte is a source of collagen essential for the rapid formation of a provisional matrix conducive for the subsequent formation of granulation tissue. Collagen synthesis also occurs by expanded populations of resident cells, under the influence of inflammatory cell-derived mediators, which results in the major accumulation of collagen during normal wound repair. However, if a chronic inflammatory state is initiated, the activated monocytes may remain in sufficient numbers to deposit collagen leading to a pathological lesion.

Basement Membrane↗